Lysis buffers for extracting nucleic acids

ABSTRACT

The present teachings relate to the extraction of nucleic acid from solid materials. Provided are useful compositions, methods, and kits for obtaining nucleic acids from a solid biological sample or an adhesive material having a biological material adherent or embedded within the adhesive substrate. The extracted nucleic acid can be used in downstream applications such as genotyping, detection, quantification, and identification of the source of the biological material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 16/013,938filed Jun. 21, 2018 (to issue as U.S. Pat. No. 10,894,957 on Jan. 19,2021), which is a Continuation of U.S. application Ser. No. 15/235,327filed Aug. 12, 2016, which is a Divisional of U.S. patent applicationSer. No. 12/882,194 filed Sep. 14, 2010 (now U.S. Pat. No. 9,447,409),which claims benefit of U.S. Provisional Application No. 61/243,136filed Sep. 16, 2009, U.S. Provisional Application No. 61/360,386 filedJun. 30, 2010, and U.S. Provisional Application No. 61/379,346 filedSep. 1, 2010, all which are hereby incorporated by reference in theirentirety.

FIELD

In general, the present teachings relate to the extraction of nucleicacid from solid materials such as bone, tooth and calcified tissues, orbiological samples embedded or adherent to adhesive materials or denimmaterials.

BACKGROUND

The extraction of nucleic acid from solid biological materials such ascalcified bone and tooth, or biological samples containing nucleic acidsembedded and/or adherent to adhesive and gum-containing materials aswell as biological materials on dried or embedded in denim materialspresents sample processing challenges and potential delays in sampleprocessing in the forensic laboratory. Forensic samples, missing person,ancient and degraded samples also have the added complication of havingPCR inhibitors potentially extracted with the eluted nucleic acid. Thepresent teachings provide useful compositions and methods for obtainingnucleic acids, such as genomic DNA and RNA, from a solid biologicalsample, an adhesive material having a biological material adherent orembedded within the adhesive substrate or a denim materials or soil. Theextracted nucleic acid can be used in downstream applications such asgenotyping, detection, quantification, and identification of the sourceof the biological material where molecular biological processes such asPCR are utilized. The lysis solutions provided can be used to preparehigh quantities of nucleic acid, such as DNA and preserve the DNAintegrity extracted from calcified tissues, or biological tissues on gumand/or adhesive substrates and materials, and denim substrates andmaterials. The solutions provide highly efficient methods for DNAextraction as well methods for the removal of PCR inhibitors and methodsto preclude extraction of PCR inhibitors. Furthermore, the procedure forextraction and purification of nucleic acids is fully automatable, usingstandard liquid handling systems.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature cited in this specification, including but notlimited to, patents, patent applications, articles, books, and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that any of the incorporated literaturecontradicts any term defined herein, this specification controls. Whilethe present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

SUMMARY OF SOME EMBODIMENTS OF THE INVENTION

In some embodiments, disclosed is a composition for lysing a solidhaving adherent or embedded within biological material. The lysisreagent solution having a composition having one or more of a detergent,a chelating agent, a reducing agent, and an enzyme. The detergent can bean anionic detergent, an ionic detergent, or a combination thereof. Thedetergent can be one or more of N-lauroyl sarcosine (NLS, also known assarcosyl or sodium lauroyl sarcosinate), sodium deoxycholate, CTAB,dodecyl β-D-maltoside, nonanoyl-N-methylglucamide, polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodium dodecyl sulfate (SDS),and combinations thereof. The chelating agent can be one or more ofethylene glycol tetraacetic acid (EGTA) and ethylene diamine tetraaceticacid (EDTA), citric acid and combinations thereof. The reducing agentcan be one or more of tris(2-carboxyethyl)phosphine (TCEP)dithioerythritol (DTE), and dithiothreitol (DTT). The enzyme can be oneor more of caspase, chymotrypsin, pepsin, proteinase K, thrombin,Staphylococcus V8 protease, pronase, papain, Bacillus sp. E1A protease,and trypsin. The salt can be one or more of sodium chloride, potassiumchloride, magnesium chloride, manganese chloride as well as fluorinatedand iodinated forms thereof. The lysis solution can have a pH rangingfrom 5.0-12.0, or 5.1, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 as well asintervals within each whole number.

In some embodiments, the solid sample lysed by the lysing solution hasnucleic acid. The solid can be a biological material, an adhesivesubstrate, or a natural or synthetic substrate. In some embodiments, thebiological material can be bone, cartilage, ligament, tendon, and tooth.In some embodiments the adhesive material can be chewing gum, cigarbutt, cigarette butt, adhesive film, adhesive label, adhesive paper,adhesive skin patch, envelope, envelope flap, stamp, e.g., a postagestamp, and adhesive tape. In some embodiments, a biological sample canbe embedded or adhered to the adhesive material.

In various embodiments, the adhesive skin patch is selected from thegroup consisting of electronic electrode, transferable tattoo,transdermal chemical substance patch and wound care dressing. And, theadhesive tape can be an adhesive bandage, athletic tape, wrapping tape,duct tape, electrical tape, hair tape, a fingerprint tape lift and soon.

In some embodiments, disclosed are methods for making a product thatcomprises a nucleic acid using the disclosed lysis solution. The methodincludes incubating a solid sample in the lysis solution and extractinga supernatant in which the nucleic acid has been extracted from thesolid sample. The solid sample can be a biological material such asbone, cartilage, ligament, tendon, and tooth, an adhesive material suchas chewing gum, cigar butt, cigarette butt, adhesive film, adhesivelabel, adhesive paper, adhesive skin patch, envelope, envelope flap,stamp, a fingerprint tape lift, and adhesive tape, a denim material. Insome embodiments, the method can also include shaking and/or vortexingthe solid sample in the lysis solution while the mixture is incubated,centrifuging the solid sample in the lysis solution after incubation,lysing the biological sample, biological material and/or solid sample inthe lysis solution to form a lysate having nucleic acid and extractingthe lysate to obtain a product with nucleic acid.

In other embodiments, disclosed are methods of separating a nucleic acidfrom a solid in which the solid is lysed by the lysis solution and thelysate is centrifuged to separate the solid from the nucleic acid whichremains in the supernatant. In some embodiments, the method can alsoinclude shaking the solid with the lysis solution and extracting thelysate to obtain a product with nucleic acid separate from the solid.

In other embodiments, the nucleic acid recovered from a fingerprint tapelift using the methods described herein can be used for identificationof the organism from which the biological material was obtained. Thenucleic acid can be used in genotyping assays for STRs, HLA markers andRFLP typing, Y-STR and Y-SNP typing, mtDNA sequencing, andinsertion/deletion polymorphisms typing. In conjunction with or separateto the genotyping assays for identification purposes the fingerprint canbe used for comparison to a fingerprint collection and/or a database offingerprints.

Other embodiments include kits for extracting nucleic acid from a solidsuch as a biological material, an adhesive material or substrate, anddenim material or substrates having epithelial, blood, semen, saliva, orother biological fluids known to one of skill in the art. The kit has atleast one of a first detergent, a chelating agent, a reducing agent, andan enzyme either as separate solutions of each or in combinations. Insome embodiments, the kit can also have optional solutions such as asolution having a polymer. The polymer comprises one or more of dextran,cellulose, cellulose derivatives, soluble starch, dextrin, cellodextrin,polyethylene glycol, heparin, glycogen, and combinations thereof. Thekit can also have a second detergent, a wash solution, a second washsolution, DNase free water, a magnetic device, and magneticallyattractable particles comprising dextran-encased magnetic nanoparticles.It should be understood that a given embodiment need not have allaspects and features described herein. It should be understood thatthese aspects and embodiments are merely exemplary and explanatory andare not restrictive of the invention.

Applicants have provided embodiments for nucleic acid extraction methodsfrom samples containing minute nucleic acid quantities. Embodiments ofsuch methods are rapid, eliminate use of strong organic solvents,simplify processing, and reduce sample handling and risks ofcross-contamination with improved user safety.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the Intercolor balance (ICB) between STRalleles for blood replicates extracted from denim. ICB was calculated bydividing maximum peak height by minimum peak height in each color(heterozygotes peaks are average and homozygotes divided by 2). Samplesare listed in Table 6.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

For the purposes of interpreting of this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). It is noted that, as usedin this specification and the appended claims, the singular forms “a,”“an,” and “the,” include plural referents unless expressly andunequivocally limited to one referent. The use of “or” means “and/or”unless stated otherwise. For illustration purposes, but not as alimitation, “X and/or Y” can mean “X” or “Y” or “X and Y”. The use of“comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.Furthermore, where the description of one or more embodiments uses theterm “comprising,” those skilled in the art would understand that, insome specific instances, the embodiment or embodiments can bealternatively described using the language “consisting essentially of”and/or “consisting of”. The term “and/or” means one or all of the listedelements or a combination of any two or more of the listed elements.

U.S. patent application Ser. Nos. 10/306,347, 11/789,352 and 60/334,029are incorporated by reference herein in their entirety for any purpose.

The practice of the present invention may employ conventional techniquesand descriptions of organic chemistry, polymer technology, molecularbiology (including recombinant techniques), cell biology, biochemistry,and immunology, which are within the skill of the art. Such conventionaltechniques include oligonucleotide synthesis, hybridization, extensionreaction, and detection of hybridization using a label. Specificillustrations of suitable techniques can be had by reference to theexample herein below. However, other equivalent conventional procedurescan, of course, also be used. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as GenomeAnalysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: ALaboratory Manual, and Molecular Cloning: A Laboratory Manual (all fromCold Spring Harbor Laboratory Press, 1989), Gait, “OligonucleotideSynthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox(2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. FreemanPub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H.Freeman Pub., New York, N.Y. all of which are herein incorporated intheir entirety by reference for all purposes.

As used herein, the term “adhere” refers to the bond between at leasttwo items or surfaces or the binding of at least two items or surfacestogether. The bond can be a fusing, gluing, or sticking together of theat least two items or surfaces.

As used herein, the term “adhesive” refers to a substance capable ofbinding at least two items. The adhesive can be a cement, glue, paste,starch, gum or chemical compound or polymer from a natural or syntheticsource and can be applied to an envelope, film, label, paper, patch,stamp, or tape as one of the at least two items. The adhesive can be aliquid or a solid or form a solid upon drying by evaporation or heating,ultra-violet light curing, or other physical means. The first item canhave the adhesive integral to it, e.g., gum or applied to it, e.g.,tape. Alternatively, the first item comprises the adhesive such that thefirst item can be bound in a removable, temporary, semi-permanent orpermanent status with respect to the bond it forms with an at leastsecond item. The adhesive can be made soluble or be made sticky by theapplication of a liquid such as saliva. For example, when a stamp islicked by a tongue prior to its application to an envelope. Anotherexample can be the flap of an envelope which when licked, such that theadhesive on the flap of the envelope becomes sticky and then when foldedover to close the envelope opening, creates a seal due to the adhesiveholding the envelope flap in constant, virtually permanent, contact withthe envelope.

As used herein, the terms “adhesive material” and “adhesive substrate”are used interchangeably and refer to items containing or have integralto its composition an adhesive, sticky surface, or adhesive composition,e.g., chewing gum, cigar butt, cigarette butt, adhesive film, adhesivelabel, adhesive paper, adhesive skin patch, envelope flap, stamp, anadhesive tape used for lifting fingerprints, biological, organic, orinorganic materials and adhesive tape.

As used herein, the term “adhesive film” refers to a thin flexible sheetthat can adhere to an item. The film can be permanently adhered to theitem or is removable.

As used herein, the term “adhesive label” or “adhesive sticker” refersto an item having identifying information or design on an upper surfacethat can adhere to another item or surface by the surface opposite theidentifying information or design. The label can permanently adhere tothe other item or is removable.

As used herein, the term “adhesive paper” refers to a paper (cellulose,wood pulp derived, vinyl, plastic, or synthetic paper) with an adherentsurface that forms a removable, temporary, semi-permanent or permanentbond with respect to the surface to which it is attached, e.g.,envelope, postage stamp, re-adherable stationary or sticky note, orsticker.

As used herein, the term “adhesive skin patch” refers to a round, oval,rectangular or other shape that can adhere in a removable fashion to anintact or damaged (wound) dermal surface, e.g., an EKG electrode, atransferable tattoo, a nicotine patch, and a plaster.

As used herein, the term “adhesive tape” refers to a flexible strip orband that can adhere to another item or surface, e.g., athletic tape,wrapping tape, Scotch® brand tape. The tape can be permanently adheredto the other item or removable. The tape can be soluble or insoluble inan aqueous or organic solution.

As used herein, the term “biological material” refers to blood, mucus,semen, saliva, skin tissue, bone, cartilage, ligament, tendon, tooth,fingerprint, etc. The biological material can be a calcified, hardened,solid and a natural composition of biological origin. The biologicalmaterial can have a solid, fluid, tissue or cells comprising nucleicacid. The biological material can include but is not limited to, nucleicacid comprising material recovered from an adhesive and/or stickymaterial including, but not limited to, chewing gum, cigar butt,cigarette butt, adhesive film, adhesive label, adhesive paper, adhesiveskin patch, envelope, envelope flap, stamp, a fingerprint tape lift, andadhesive tape.

As used herein, the term “biological sample” refers to a biologicalmaterial adherent to or imbedded in an adhesive material, e.g., blood,mucus, semen, saliva, skin cell, skin tissue, bone, cartilage, ligament,tendon, tooth, a fluid, tissue, or cells. The biological samplecomprises nucleic acid.

As used herein, “DNA” refers to deoxyribonucleic acid in its variousforms as understood in the art, such as genomic DNA, cDNA, isolatednucleic acid molecules, vector DNA, and chromosomal DNA. “Nucleic acid”refers to the nucleic acid molecule or molecules, DNA or RNA(ribonucleic acid) in any form. As used herein, the term “nucleic acidmolecule” or “extracted nucleic acid” refers to a nucleic acid molecule(DNA or RNA of any form) that has been recovered from its nativeenvironment. Some examples of extracted nucleic acid molecules arepartially or substantially purified nucleic acid molecules, nucleicacids obtained from forensic and other samples comprising biologicalmaterial, such as blood, mucus, semen, saliva, skin tissue, bone,cartilage, ligament, tendon, and tooth, etc. Also envisioned isbiological material recovered from an adhesive and/or sticky materialincluding but not limited to, chewing gum, cigar butt, cigarette butt,adhesive film, adhesive label, adhesive paper, adhesive skin patch,envelope, stamp, and adhesive tape, as well as in nucleic acid samplesderived from archeological, crime scene processing, forensic, humanidentification, natural and mass disaster scenes collected by forensic,crime, research, search and rescue, and recovery methods and techniques.

As used herein, the terms “fingerprint” and “fingerprint image” are usedinterchangeably herein and refer to friction skin ridge impressions madewhen the epidermal ridges and indentations of the skin found on aventral surface of a digit, including but not limited to, fingers andtoes, a thumb, as well as a palm and a sole of a foot, contact asurface.

As used herein, the term “genomic DNA” refers to the nucleic acidscomprising the chromosomal DNA sequence of a gene or segment of a gene,including the DNA sequence of noncoding as well as coding regions.Genomic DNA also refers to DNA isolated directly from biological samplese.g., bone, tooth, tissue, cells or chromosomes or the cloned copies ofall or part of such DNA. Genomic DNA also refers to DNA extracted froman adhesive or sticky material e.g., chewing gum, cigar butt, cigarettebutt, adhesive film, adhesive label, adhesive paper, adhesive skinpatch, envelope, stamp, and adhesive tape. Gum can be of natural orsynthetic origin. Natural gum can be latex, gum Acacia, Guar gum, Chicleand Chicle varieties such as chicoo and chicozapote. Synthetic gum canbe polyisobutylene.

As used herein, the terms “denim material” and “denim substrate” areused interchangeably and refer to materials of either animal orvegetable origin. The denim material composed entirely of cotton orcotton blended with natural or synthetic fibers. The cotton denim fabriccan include but is not limited to blue, dark blue, black, stone-washed,acid washed and the like.

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably herein and refer tosingle-stranded and double-stranded polymers of nucleotide monomers,including without limitation 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H+, NH4+, trialkylammonium, Mg2+, Na+, and the like. A polynucleotidemay be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof and can include nucleotideanalogs. The nucleotide monomer units may comprise any nucleotide ornucleotide analog. Polynucleotides typically range in size from a fewmonomeric units, e.g., 5-40 when they are sometimes referred to in theart as oligonucleotides, to several thousands of monomeric nucleotideunits. Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U” denotes deoxyuridine, unless otherwise noted.

As used herein, the term “solid” refers to a material that is not aliquid such as an adhesive containing material as well as hardened,dense materials having a matrix such as calcified biological materialse.g., bone, tooth, as well as cartilage, tendon and ligament and commondust which is composed of sloughed skin cells. The solid can contain oris suspected of containing a nucleic acid.

As used herein, the term “solid sample” refers to a hard, densematerial, likely a biological material such as bone, cartilage,ligament, tendon and tooth or an adhesive material that is nonporousand/or water insoluble and contains or is suspected of containing anucleic acid.

Also herein, the recitations of numerical ranges and numerical ranges byendpoints include all numbers subsumed within that range (e.g., 1 to 5includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In some embodiments of the present teachings, compositions, methods, andkits are described in which nucleic acid molecules can be separatedand/or extracted from samples and, in some embodiments, in which theproduct made from the compositions, methods and kits are nucleic acids.In some embodiments, the compositions, methods, and kits of the presentteachings result in the formation of a product which comprises theextracted nucleic acid.

In some embodiments of the present teachings, compositions are providedwherein a sample can be treated with a lysis solution comprising atleast one each of a detergent, a chelating agent, a reducing agent, andan enzyme, and combinations thereof in order to extract nucleic acidmolecules from the sample. In various embodiments, the sample cancomprise one or more of free nucleic acids; extracted from solidmatrices including, but not limited to, biological materials such asbone, cartilage, ligament, tendon, and tooth, and so on. In variousembodiments, the sample can comprise nucleic acid extracted fromadhesive materials, including, but not limited to, materials such aschewing gum, cigar butt, cigarette butt, adhesive film, adhesive label,adhesive paper, adhesive skin patch, envelope flap, stamp, a fingerprinttape lift, and adhesive tape. In some embodiments, the sample cancomprise nucleic acid extracted from denim materials including, but notlimited to, denim fabric including blue, dark blue, black, stone-washed,acid washed and the like. In additional embodiments of the presentteachings, methods are described wherein a nucleic acid can be extractedfrom a sample, comprising the steps of incubating the sample with asolution comprising one or more of a detergent, a chelating agent, areducing agent, a salt, and an enzyme; and extracting the supernatant.The incubation can be at room temperature, 35° C., 40° C., 41° C., 42°C., 43° C., 44° C., 45° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55°C., 56° C., 57° C., 58° C., 59 ° C., 60° C., 65° C. and 70° C., as wellas intervals therein. Such methods may further comprise the steps ofcentrifuging the incubation solution to remove solids from thesupernatant and treating the supernatant to isolate the nucleic acids.

The nucleic acids thus obtained can then be utilized in any of variousdownstream applications and analyses such as, for example,quantification, detection, and genotyping of specific nucleic acids oreven of a biological species. These analyses can be performed, forexample, by PCR amplification. As one example, in forensic DNA analysisthe human nuclear DNA (nDNA) and/or genomic DNA can be obtained fromcomplex biomaterials and then genotyped using PCR. As another example, aDNA preparation can be used for quantification of human DNA, or morespecifically human male DNA, using real-time PCR systems such asQuantifiler® or Quantifiler® Duo kits (Applied Biosystems/LifeTechnologies Corp., Foster City, Calif.), and/or genotyped for autosomalor Y-chromosomal short tandem repeat loci using systems such as, forexample, AmpFiSTR® kits. Based upon the amount of DNA present in asample, a particular genotyping system can be selected that will yieldthe best results for the particular analysis required. Therefore, inorder to best utilize nucleic acids in downstream applications, it isparticularly desirable that the extraction and isolation methods resultnot only in a product of high yield, but also one that is relativelyfree of inhibitors of downstream applications such as PCR. The nucleicacids analyzed by quantification, detection, and genotyping can be usedin the identification of the source of the nucleic acid.

Typically, forensic evidence samples are variable in sample types,substrate, or matrix wherein the biological materials are embedded,including but not limited to, solid biological samples or the surfaceupon which the forensic sample was collected, which can result in sampleexposure to environmental insults, contamination by PCR inhibitors andlimit the quantity of starting sample material. Extraction of nucleicacid, including, but not limited to, DNA from forensic evidence samples,often exposed to temperature variation, excessive moisture ordehydrating conditions, and sample contaminants as would be known to oneof ordinary skill in the art during acquisition and processing, can leadto difficulties in isolation of nucleic acid and the potential ofcontamination with compounds, e.g., dye or other chemical treatments ofdenim fabric that act to inhibit PCR, and which therefore interfere withattempts at genotyping or other analyses. It is desirable to improveextraction processes and in so doing, remove inhibitors during theisolation of nucleic acid, such as DNA or RNA for use in forensicanalysis, prior to use in subsequent processes employing enzymes, suchas, amplification.

As an example, forensic samples can employ adhesive materials in samplecollection or be a component of the sample itself. The presence of theadhesive often decreases the overall recovery of nucleic acid as theadhesive can interfere with the isolation process as when particulatesare used to isolate the nucleic acid. In examples present below it wasshown that nucleic acid extracted from cigarette butts, chewing gum, anddried blood, saliva, and biological material (including skin cells andcells from a fingerprint) in a tape lift resulted in higher nucleic acidrecoveries and was free of PCR contaminants when profiled by shorttandem repeat (STR) analysis.

Another example of difficult forensic samples are solid biologicalmaterials including, but not limited to, calcified tissues such as boneand tooth. The dense matrix requires lengthy, labor intensive processes,often lasting more than 24 hours, before minute amounts of nucleic acidare extracted. The disclosed teachings achieved maximum recovery of DNAby disruption of the calcified matrix so that the majority of thebiological material is exposed to the lysis reagent solution. The yieldof DNA from bone and tooth samples is summarized in Table 1. In examplesto follow the time required to extract nucleic acid from bone and toothat optimum yield was achieved after only two hours. Because thedisclosed lysis solution compositions and methods for extracting nucleicacid results in improved and faster DNA recovery, the amount of DNApresent in a sample can be detected with greater sensitivity byreal-time PCR methods known to one of skill in the art.

Various embodiments of the present teachings relate to efficientextraction of nucleic acids such as, for example, genomic DNA in such aquantity that subsequent isolation and elution of the nucleic acid usingthe PREPFILER™ Forensics DNA Extraction Kit (P/N 4392852 or 4392353,Applied Biosystems, Foster City, Calif.) provides nucleic acid suitablefor DNA quantification and analyses. Embodiments of these teachings thusenable effective extraction of nucleic acids, such as genomic DNA, fromvarious solid types of biological materials, adhesive comprisingmaterials as well as biological fluid samples from denim. In addition,nucleic acids such as genomic DNA can be isolated from either small orlarge quantities of the biological materials that are commonly processedin laboratories such as, for example, those involved in genotypinganalyses.

Embodiments of the lysis solution provided herein can comprise a mixtureof a chelating agent (preferably ranging from 250-500 mM), such asethylene glycol tetraacetic acid (EGTA) and/or ethylene diaminetetraacetic acid (EDTA) and/or citric acid; detergents (preferablyranging from 0.1% to 3%), such as at least one of N-lauroyl sarcosine,sodium deoxycholate, CTAB, N-dodecyl β-D-maltoside,nonanoyl-N-methylglucamide, Triton® X-100, NP-40, and/or sodium dodecylsulfate; a salt (ranging from 50 mM to 500 mM), such as sodium chloride,potassium chloride, magnesium chloride, manganese chloride as well asfluorinated and iodinated forms thereof; and reducing agent (preferablyranging from 10 to 100 mM), such as tris(2-carboxyethyl)phosphine (TCEP)dithioerythritol (DTE), and dithiothreitol (DTT), and an enzyme(preferably ranging from 0.5 to 1 mg/mL), such as at least one ofcaspase, chymotrypsin, pepsin, proteinase K, thrombin, Staphylococcus V8protease, pronase, papain, Bacillus sp. E1A protease, and trypsin.Tables provided below indicate the extraction efficiency as a comparisonof various incubation times in the lysis solution, quantity, and type ofstarting solid, adhesive or denim material. The lysis solution extractsthe nucleic acid from the solid matrices or adhesive material such thatthe nucleic acid supernatant can be isolated and cleansed of impuritiesusing the PrepFiler™ Kit.

These embodiments and other features of the present teachings willbecome more apparent from the description herein.

Various embodiments of the present teachings relate to a lysis solutionand methods of using the solution, amenable to assembly in a kit, forthe extraction of nucleic acids including, but not limited to, genomicDNA, from adhesive materials containing biological samples and solidsamples, such as biological samples, thought to contain or known tocomprise nucleic acid. The adhesive material can be for example, a tapelift collected by forensic methods, e.g., a dried blood, saliva, andfingerprint, as well as an envelope flap, stamp, including but notlimited to a postage stamp, trading stamp, hunting license stamp,conservation stamp, chewing gum, cigar butt, cigarette butt, adhesivefilm, adhesive label, adhesive paper, adhesive skin patch, envelope,stamp, and adhesive tape and such. The adhesive material is thought tocontain nucleic acid attached or embedded in the adhesive material andthe present teachings relate to the extraction of the nucleic acid, ifpresent, from the adhesive material sample. Adhesive material has beenshown to interfere with subsequent nucleic acid isolation, purificationand overall yield when using the PrepFiler™ kit. The adhesive interfereswith the handling of the magnetic particles, in the polymer-nucleicacid-magnetic particle complex formed in the PrepFiler protocol. Theformation of the complex is such that the polymer entraps the nucleicacid, polymer attaches to magnetic particles, and so indirectly connectsthe nucleic acid with the magnetic particles until the complex is washedto remove contaminants and inhibitors such that the nucleic acid isamendable to use in downstream applications, such as PCR. Obtainingnucleic acid from the adhesive containing sample was difficult, tediousand of low yield prior to the development of the lysis solution taughtherein.

The dyes, pigments and fibers found in denim fabric as is used in themanufacture, including, but not limited to blue jeans, skirts, andjackets, has been found to inhibit STR analysis of nucleic acidsisolated therefrom in genotyping assays such as the Identifiler® kit(Applied Biosystems). The fibers appear to complex with the pigments ordyes and interferes with the handling of the magnetic particles, in thepolymer-nucleic acid-magnetic particle complex formed in the PrepFilerprotocol. This has made obtaining nucleic acid from the denim samplecompromised by PCR inhibitors, DNA recovery is difficult, tedious and oflow yield prior to the development of the Tris-Cl lysis solution taughtherein.

Various embodiments of the present teachings relate to a nucleic acidextraction, such as for genomic DNA, comprising lysis solution, methods,and kits for extraction of the nucleic acids from solid biologicalsamples, including, but not limited to, bone, cartilage, ligament,tendon, and tooth, and adhesive. Embodiments of these methods cancomprise: cleaning the solid sample with a mild detergent solution,air-drying overnight, removing the exterior layer with a sanding stone,and pulverizing the sample. The prepared sample is then incubated withlysis solution such that the nucleic acid containing cells are releasedfrom the solid matrix. The solid biological material contains nucleicacid, and the present teachings relate to the extraction of the nucleicacid from the solid material. Calcified and hardened biologicalmaterials have been shown to provide low yields of nucleic acid whenusing the PrepFiler™ kit (Applied Biosystems, Foster City, Calif.),because the solid matrices provide unique challenges for the extractionand isolation of nucleic acid. The yield of nucleic acid from the solidbiological material has been improved by use of the lysis solutiontaught herein prior to the PrepFiler protocol.

Standard nucleic acid isolation techniques, including cell lysis, andwashing and elution of nucleic acids, are well known in the art andunless otherwise noted, can be carried out according to varioustechniques as described, for example, in DNA Typing Protocols: MolecularBiology and Forensic Analysis, 1st edition, B. Budowle et al., eds.,Eaton Publishing Co. (2000); JM Butler, Forensic DNA Typing: Biology,Technology, and Genetics of STR Markers, 2nd edition, Elsevier AcademicPress (2005); or P. Gill, “Application of Low Copy Number DNAProfiling,” Croatian Medical Journal42:229-232 (2001); FR Bieber et al.,“Isolation of DNA from Forensic Evidence,” Current Protocols in HumanGenetics, Supplement 26 (2000); Forensic DNA Profiling Protocols,Methods in Molecular Biology, vol. 98, P J Lincoln and J. Thomson, eds.,Humana Press (1998).

Applicants have discovered that nucleic acids are more easily isolatedand purified when using the PrepFiler protocol for nucleic acidcontaining adhesive materials and solid biological samples by using thelysis reagent solution taught herein having one or more of a detergent,a chelating agent, a reducing agent, a salt, and an enzyme. The lysissolution improves the efficiency and yield of nucleic acids, such asgenomic DNA, from adhesive materials, solid biological samples. Thedetergent can be ionic or anionic. Some examples of appropriatedetergents include, but are not limited to, N-lauroyl sarcosine (NLS),lauroyl sarcosinate, also known as sarcosyl, an ionic surfactant derivedfrom sarcosine; hexadecyltrimethylammonium bromide orcetyltrimethylammonium bromide (CTAB); deoxycholate; sodium citrate;sodium deoxycholate; dodecyl β-D-maltoside; nonanoyl-N-methylglucamide;sodium dodecyl sulfate; polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether (commercially known as Triton®X-100); and combinations thereof. In some embodiments, the detergentcomprises NLS in the range of 0.5 to 3% w/v. Some examples ofappropriate chelating agents are, but not limited to, ethylene diaminetetraacetic acid (EDTA); ethyleneglycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and citricacid in the range of about 250 to 750 mM. Examples of appropriatereducing agents include but are not limited to,tris(2-carboxyethyl)phosphine (TCEP) dithioerythritol (DTE), anddithiothreitol (DTT) in the range of about 10 to 100 mM, prepared freshprior to use. Some examples of appropriate enzymes are, but not limitedto, caspase, chymotrypsin, pepsin, proteinase K, thrombin,Staphylococcus V8 protease, pronase, papain, Bacillus sp. E1A protease,and trypsin in the range of 0.5 to 10 mg/ml final concentration in thelysis solution. In some embodiments the lysis solution is prepared justbefore use by combining the chelating agent, at a pH of about 7.5 to 8.5with detergent and salt, followed by adding the reducing agent solutionand the enzyme.

In some embodiments, the chelating agent is prepared in a solutionhaving the detergent with or without the salt, the reducing agent isfreshly prepared, and the enzyme is in solution. The salt is either insolution or present in the detergent solution. The solutions can then becombined just prior to use to form the lysis solution. Alternatively, insome embodiments of the present teachings a lysis solution can beprepared and then freeze-dried. The freeze-dried lysis reagent can thenbe solubilized just prior to use. Such methods of freeze-drying areconventional and known to one of ordinary skill in the art. The resultof a freshly prepared lysis solution is that the components have notdegraded or oxidized such as may occur with prolonged storage orexposure to light and therefore decrease nucleic acid extraction andoverall yield of nucleic acid. In some embodiments, the adhesivematerial or solid biological material is incubated at from about 50° C.to 70° C. for two hours to release cellular material from adhesivematerials or solid biological matrices with 56° C. being a commontemperature used. In some embodiments, the natural or synthetic materialis incubated at from about 40° C. to 70° C. for 40 min to 1 hour. Theextraction can be assisted by shaking. In some embodiments the lysissolution further comprises compounds to protect the released nucleicacids and maintain their compatibility for use in downstream assays suchas, for example, PCR assays, and in particular DNA genotyping systems,for example STR assays.

Following incubation of the adhesive material, or solid biologicalsample, the sample can be centrifuged to pellet residual solid tissue,or adhesive material. The resulting supernatant product comprisesnucleic acids. The supernatant is suitable for nucleic acid isolationand purification using methods known to one of ordinary skill in the artor commercial nucleic acid isolation and purification products such asthe PrepFiler™ Kit. The kit can be used to remove cellular debris,residual contaminants and/or inhibitors of PCR.

In some embodiments of the present teachings, then, nucleic acidmolecules can be isolated from solid matrices including biologicalsamples including but not limited to: bone; cartilage; ligament; tendon;and tooth. In some embodiments, the biological sample is calcified,mummified, baked or otherwise in a natural or preserved state. In someembodiments, the solid biological sample is fragmented, crushed,pulverized, or otherwise ground into a fine particulate, such as apowder. The yield of nucleic acid, such as DNA, is variable dependingupon the age of the solid material, the amount of starting material andthe degradation or preservation of the sample. The yield of the nucleicacid as DNA from bone and tooth samples is summarized in Table 1.

The yield of DNA from 100 mg of long bone samples ranged from 0.25 to2.15 ng, which is typical of such a sample type since each bone sampleis unique in the content and state of biological material. The CT valuesfor internal PCR control (IPC) determined by the Quantifiler® Human DNAQuantification Kit (Applied Biosystems) for the bone samples tested didnot increase significantly compared to that for the non-template control(NTC, Table 1). If the DNA extract contained PCR inhibitors, typicallyan upward shift in CT value for the sample compared to the IPC CT valuefor the NTC would be expected (Applied Biosystems, Quantifiler® HumanDNA Quantification Kit and Quantifiler® Y Human Male DNA QuantificationKit. User's Manual. PN 4344790 Rev. B, (2003)). Thus, the PCR inhibitorspresent in bone and tooth samples have been efficiently removed duringthe extraction of DNA using the lysis reagent solution followed byisolation and purification with the PrepFilerTM kit reagents.

TABLE 1 Yield of human DNA from bone and tooth Total Yield IPC C_(T)Sample^(a) Description (ng) Value Bone-1 100 mg long bone 1.9 27.05Bone-2 100 mg long bone 0.66 27.12 Bone-3 100 mg long bone 1.19 26.96Bone-4 100 mg long bone 1.6 27.19 Bone-5 100 mg long bone 0.8 27.12Bone-6 100 mg long bone 0.24 27.11 Bone-7 100 mg long bone 2.22 27.17Bone-CA 50 mg clavicle bone 7.95 ± 1.2 27.012 Tooth 10 mg 450 ± 50 26.95Extraction Blank 0 26.80 Non-Template Control (NTC) NA^(b) NA^(b)^(a)Bone samples were of unknown age. Tooth sample was 1 year old.^(b)Not applicable.

The quantity of bone sample tested for optimum extraction and yield ofnucleic acid varied from 50 mg to 200 mg and the volume of the lysissolution supernatant collected was adjusted, whenever necessary, to 200μL using PrepFiler™ lysis buffer prior to isolation of DNA. The totalyield of DNA was almost doubled when the quantity of bone powder wasincreased from 25 to 50 mg powder (Table 2). Further increase in thequantity of bone powder increased the yield of DNA but the incrementalincrease in the yield of DNA was not proportional to the quantity ofbone powder. The evaluation was conducted in triplicate. The volume oflysis solution supernatant recovered after incubation of 200 mg of bonepowder was 130 μL compared to 200 μL from 50 mg of bone powder.Therefore, processing of bone powder greater than 200 mg did notappreciably increase DNA yield.

TABLE 2 Bone-CA Total Yield Yield/g IPC C_(T) (mg) (ng) bone Value 25 4.8 ± 1 0.2 27.05 50  9.3 ± 3 0.18 26.94 100 12.0 ± 3 0.12 27.03 20021.6 ± 6 0.11 26.89 Extraction Blank 0 NA NA Non-Template Control (NTC)NA NA 26.85

The extent of extraction of nucleic acid material from bone samples wasinvestigated at different time intervals from 1 to 18 hours. 50 mg ofbone was extracted in triplicate. The yield of DNA is summarized inTable 3. The yield of DNA increased by 1.54-fold when the time forextraction of the bone sample with the lysis solution was increased from1 to 2 hours. However, further increase in the incubation time to 18hours did not increase the yield of DNA substantially. However,incubation time up to 18 hours did not affect the extraction adversely.

TABLE 3 Time for lysis Total Yield IPC C_(T) (hours) (ng) Value 1 5.2 ±1   27.132 2 8.0 ± 1   27.047 4 8.5 ± 1.5 26.952 8 8.8 ± 1.5 26.913 1811.4 ± 1   26.923 Non-Template Control (NTC) NA 26.815

DNA extracts from seven long bone samples (bone 1 through 7) wereprocessed for STR analysis using AmpFiSTR® Identifiler® and MiniFiler™kits (Applied Biosystems), following the manufactures protocol. Theresults are summarized in Table 4. The Identifiler Kits amplifies atotal of 16 STR loci and the MiniFiler kit amplifies 9 STR loci. In eachsample tested, conclusive loci are compared to the total amplifiableloci for each of the kits. Bone-4 sample provided a complete STR profileusing both kits. Bone-6 did not provide results when amplified using theIdentifiler® kit but provided amplification of only 4 loci using theMiniFiler™ kit. All other bone samples tested provided complete STRprofiles with the MiniFiler™ kit and partial STR profiles with theIdentifiler® kit. The MiniFiler™ kit is designed to obtain profiles fromdegraded DNA samples (J. J. Mulero, et al., J. Forensic Sci. (2008)53:838-852). The results indicate that the bone samples were aged anddegraded to different extent.

TABLE 4 Sample^(a) Identifiler ® Kit MiniFiler ™ Kit Bone-1  9/16 9/9Bone-2  9/16 9/9 Bone-3 10/16 9/9 Bone-4 16/16 9/9 Bone-5  9/16 9/9Bone-6  0/16 4/9 Bone-7 15/16 9/9 ^(a)Bone samples were of unknown age.

In some embodiments, the present teachings include using a plurality ofsamples ranging from when only trace amounts of biological material ispotentially present. The sample amount can range from 50 to 200 mg ofcalcified tissue or solid biological sample in each tube and then thesupernatants are combined and concentrating by standard methods prior toisolation and purification.

In some embodiments of the present teachings, then, nucleic acidmolecules can be isolated from adhesive materials, including, but notlimited to: chewing gum; cigar butt; cigarette butt; adhesive film;adhesive label; adhesive paper; adhesive skin patch; envelope flap,stamp, and dried blood, dried saliva, environmental sampling, includingbut not limited to dust, soil, dirt and a fingerprint via an adhesivetape lift, and adhesive tape. In some embodiments, the adhesive skinpatch includes, but is not limited to: EEG or EKG electronic electrode;transferable tattoo; transdermal chemical substance patch and wound caredressing. In some embodiments, the adhesive tape includes, but is notlimited to: tape; athletic tape; forensic tape; and so on. In someembodiments, the adhesive paper includes, but is not limited toenvelope, removable paper, stamp and so on.

Comparisons of DNA yields from samples containing adhesives such ascigarette butts, chewing gum, tape lifts, envelopes, and stamps usingthe disclosed lysis reagent solution, PrepFiler™ lysis buffer and theEZ1 DNA Investigator Kit (Qiagen, Valencia, Calif.) are shown in Table5. In general, the yields of DNA using the disclosed lysis reagentsolution were higher for tape lift and cigarette butt samples comparedto the yields obtained using the PrepFiler™ lysis buffer. Similarly, theyield of DNA for two of the three chewing gum samples tested was higherusing the disclosed lysis reagent solution. Samples were generally ofsimilar quantities of biological material (see Examples I and IV).Because the evaluations involved using real-world sample types as wouldbe encountered in forensics laboratories, the sample types were notabsolutely uniform. Thus, the resulting yields of DNA are useful as ameasure of the overall performance of the lysis reagent solution. It hasbeen observed that the tape lift and chewing gum samples each release alarge amount of adhesive when the PrepFiler lysis buffer was used. Forthe tape lift samples, some of the clumps of the adhesive could beremoved before further PrepFiler protocol processing however, in many ofthe samples, the magnetic particles did not form a pellet and some ofthe samples had to be centrifuged during PrepFiler protocol wash steps.The lysis reagent solution had a much smaller amount of the adhesivereleased during the extraction process. Consequently, the magneticparticles formed stronger pellets during the subsequent PrepFiler washsteps leading to improved nucleic acid recovery, including DNA yields.Small pieces of paper were removed by centrifugation from the cigarettesamples. The Sherman brand cigarette paper is purple, and it wasobserved that the purple dye was released under the PrepFiler™ lysisconditions. Additionally, the IPC C_(T) values for all case type samplesstudied were similar to that obtained for NTC indicating effectiveremoval of PCR inhibitors from the biological samples and substrates(Table 5).

TABLE 5 STR profile results from long bone samples^(a) Lysis ReagentSoln. PrepFiler ™ Lysis Total DNA Total DNA Substrate Sample Yield(ng)^(a) IPC C_(T) Yield (ng) IPC C_(T) Tape lifts Tape + 2 μL blood^(b)40 26.6 14 27.4 Tape lift of 2 μL 20 27.3 17 27 blood Tape + 5 μLsaliva^(b) 143 27.1 64 26.8 Tape lift of 5 μL 7 26.9 3 27.4 saliva Tapelift, shirt cuff 0.6 26.6 ND^(c) ND Tape lift, shirt cuff 4 26.7 ND NDTape lift, shirt collar 2.5 26.8 ND ND Tape lift, shirt collar 0.6 26.9ND ND Substrate Blank 0 26.8 0 26.8 Extraction Blank 0 27.0 0 27.0Chewing Winterfresh 7 27.0 15 26.4 Gum^(d) Doublemint 6 27.1 3 27.3Orbit 20 27.1 19 26.6 Extra White 27 28.1 ND ND Airwave (2 min. 7 27.8ND ND chewed) Airwave (40 min. 30 27.7 ND ND chewed) Blast Mentos 3027.7 ND ND Berry-Lime Extraction Blank 0 27.1 0 27.1 Cigarette Marlboro174 26.7 25 27.6 Butts Marlboro Menthol 75 26.7 39 27.1 Light Sherman424 26.6 111 29.0 Gauloises Menthol 76 26.7 22 28.0 Marlboro Menthol 3526.7 3 26.8 Light^(e) Holiday Gold 12 26.9 ND ND Holiday Menthol 37 26.5ND ND Substrate Blank 0 26.7 0 26.7 Extraction Blank 0 26.7 0 26.7^(a)Elution volume was 50 μL. The quantities presented are averagevalues. ^(b)2 μL of blood or 5 μL of saliva was added directly to the1.5 cm × 2.5 cm piece of tape. ^(c)Not determined ^(d)⅛ of a piece ofgum processed, all are Wrigley's brand except Blast Mentos Berry-Lime^(e)Weathered sample-left outside for 72 hr before analysis. 72 hrtemperature range was 4-15° C.

In some embodiments of the present teachings, a Tris-Cl Lysis Solutioncan used to isolate nucleic acid molecules from denim materialscontaining on the surface or embedded within a biological fluid sampleincluding but not limited to, for example, hair, feces, blood, tissue,urine, saliva, cheek cells, vaginal cells, skin, for example skin cellscontained in fingerprints, buccal sample, amniotic fluid containingplacental cells, and amniotic fluid containing fetal cells, and semen.The biological fluid can be moist, wet, dried, or adherent to the denimmaterial. In some embodiments, the biological fluid sample can be foundwithin an environmental sample such as soil. In other embodiments,disclosed are methods for extracting a biological fluid material fromdenim material simultaneously with lysing the cells within thebiological fluid material and eluting the nucleic acid from thesubstrate. In some embodiments, the method can also include heating orincubating a Tris-Cl lysis solution with the denim substrate containingthe biological fluid material. In some embodiments, the Tris-Cl lysissolution is centrifuged to separate the nucleic acid containing lysatefrom the substrate.

In some embodiments of the present teachings, compositions are providedwherein a sample can be treated with a Tris-Cl lysis solution comprisingTris-chloride (Tris-Cl), at least one each of a detergent, a chelatingagent, a reducing agent, and an enzyme, and combinations thereof inorder to extract nucleic acid molecules from the denim substrate sample.The detergent can be one or more of N-lauroyl sarcosine, sodiumdeoxycholate, CTAB, dodecyl β-D-maltoside, nonanoyl-N-methylglucamide,polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodiumdodecyl sulfate (SDS), and combinations thereof. The chelating agent canbe one or more of ethylene glycol tetraacetic acid (EGTA) and ethylenediamine tetraacetic acid (EDTA), citric acid and combinations thereof.The reducing agent can be one or more of tris(2-carboxyethyl)phosphine(TCEP) dithioerythritol (DTE), and dithiothreitol (DTT). The enzyme canbe one or more of caspase, chymotrypsin, pepsin, proteinase K, thrombin,Staphylococcus V8 protease, pronase, papain, Bacillus sp. E1A protease,and trypsin. The salt can be one or more of sodium chloride, potassiumchloride, magnesium chloride, manganise chloride as well as fluorinatedand iodinated forms thereof. The lysis solution can have a pH rangingfrom 5.0-12.0, or 5.1, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 as well asintervals within each whole number.

In some embodiments, the sample can comprise nucleic acid extracted fromdenim materials including, but not limited to, denim fabric includingblue, dark blue, black, stone-washed, acid washed and the like. Inadditional embodiments of the present teachings, methods are describedwherein a nucleic acid can be extracted from a sample, comprising thesteps of incubating the sample with a solution comprising Tris-Cl, oneor more of a detergent, a chelating agent, a reducing agent, a salt, andan enzyme; and extracting the supernatant. The incubation can be at roomtemperature, 35° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 65° C. and 70° C., as well as intervals therein. Suchmethods may further comprise the steps of centrifuging the incubationsolution to remove solids from the supernatant and treating thesupernatant to isolate the nucleic acids.

In some embodiments, inhibitors of PCR can be extracted or released intothe lysate or eluate during the process of nucleic acid extractionresulting in poor quality DNA. The quality of the nucleic acid extractedor recovered from a biological fluid sample can adversely impactanalysis of the nucleic acid in processes such as, for example,quantification, detection, and genotyping of specific nucleic acids or abiological species.

In some embodiments, the Tris-Cl can be at a concentration of from 10 mMto 600 mM and at a pH of from 5 to 12. In some embodiments, thedetergent can be present at from 0.1% to 5.0% and the chelating agentcan be present at from about 1.0 mM to 600 mM at a pH of from 7.0 to 8.5(adjusting pH with 0.5-1M NaOH). Some examples of appropriate saltsinclude, but are not limited to, sodium chloride, potassium chloride,magnesium chloride, manganese chloride as well as fluorinated andiodinated forms thereof in the range of about 1 mM to 1 M. In someembodiments, the enzyme concentration is in the range of 1 mg/uL to 50mg/mL and the reducing agent concentration is in the range of 10 mM to 1M.

Various types, weights, and colors of denim fabric (obtained fromJo-Ann's Fabrics, San Mateo, Calif.) were used to analyze the Tris-Cllysis reagent solutions. Basically, blood was applied to each denim typeand a 5 mm denim punch was removed from the center of the blood spot,approximating 14 of dried blood. Comparisons of Intercolor Balance (ICB)using the Identifiler® kit (Applied Biosystems) on DNA recovered fromthe denim samples listed in Table 6, some of which contained indigo dye,acid wash treatment, stone wash treatment, ice wash treatment, blackdye, texture, printing and of varying weights and finishes is disclosedin FIG. 1 following using nucleic acid extraction with the disclosedTris-Cl-buffered lysis reagent solution, PrepFiler™ lysis buffer andIdentifiler® kit (Applied Biosystems). In general, the DNA quality usingthe disclosed Tris-Cl-buffered lysis reagent solution were sufficient toobtained full STR profiles for each sample tested. These results showthat the disclosed Tris-Cl-buffered lysis reagent solution can providehigh quality nucleic acid even in the presence of indigo, black dye, andchemical treatments, known PCR inhibitors.

TABLE 6 Denim ID Description 1 10 OZ Distressed STR 2 10.5 OZ DK bluestove 3 5.8 OZ sun-bleached DNM 4 9 OZ DK blue stovepipe 5 8 OZ bluestovepipe D 6 4 OZ DK blue denim 7 8 OZ DK blue denim 8 8 OZ indigostovepipe 9 9.9 OZ blue textured st 10 11 OZ black STR denim 11 5 OZblue xhatch denim 12 12. L BL ice wsh 13 8 OZ dirty xhatch denim 14 10OZ indigo wash denim 15 10 OZ LT blue SIR sto 16 10.5 OZ med blue xhat17 9.75 OZ lt blue stove 18 7 OZ blue STR stovepipe 19 10 OZ sbl washdenim 20 5 OZ blue xhatch denim 21 9 OZ blue textured st 22 9 OZ medblue str denim 24 6.5 OZ indigo denim 25 7 OZ lt blue slub denim 27 sblwsh denim 10 OZ 28 28. ind brush denim 29 10 OZ distressed nost 30 12.5OZ nostalgic str 31 4 OZ lt blue denim 32 11 OZ lt blue str denim 34 4OZ lt blue denim 36 7 OZ blue denim 37 9 OZ indigo shine was 38 7 OZblue str stovepipe

In some embodiments of the present teachings, then, nucleic acidmolecules can be isolated from denim using Tris-Cl-buffered lysisreagent solution.

A capillary electrophoresis electron micrograph of STR profiles obtainedfrom bone, tooth and some adhesive containing materials were typical ofsuch sample types. Furthermore, the STR profiles demonstrated that theDNA obtained was free of any PCR inhibitors (data not shown, Stray etal., Forensic Science International: Genetics Supplement Series 2 (2009)159-160, incorporated by reference herein). The lysis reagent solutionwas shown to disrupt calcified tissue matrices, achieve effective DNAextraction from pulverized bone and tooth samples as well as fromadhesive materials such as tape, chewing gum, envelope, stamp, andcigarette butts. The results for denim materials for the STR profilesdemonstrated that the DNA obtained was free of any PCR inhibitors (datanot shown). The Tris-Cl-buffered lysis reagent solution was shown toachieve effective DNA extraction from denim samples. Both the lysisreagent solution and the Tris-Cl-buffered lysis reagent solutionresulted in improved recovery of nucleic acid, improved DNA isolation,purification, and the DNA was absent interfering materials andimpurities that would compromise or impede further analyses such as PCR,real-time PCR and STR profiling.

In other embodiments of the present teachings, the nucleic acidrecovered from a fingerprint using the methods described herein can beused for identification of the organism from which the biologicalmaterial or biological sample containing the nucleic acid was obtainedas discussed supra. In conjunction with or separate to the nucleic acididentification assays the fingerprint image can be used for comparisonto a forensic database including but not limited to a fingerprintcollection and/or a database of fingerprints. Fingerprints are awell-established forensic identification tool and numerous fingerprintdatabases and collections exist worldwide for purposes of identificationand confirmation/verification of identity. The advantage of utilizingnucleic acid obtained from a fingerprint provides further confirmatoryand conclusive evidence of identification and further, association of anidentified individual with the surface and so the place or object fromwhich the fingerprint nucleic acid was obtained.

In other embodiments of the present teaching, the nucleic acid recoveredfrom denim using the methods described herein can be used foridentification of the organism from which the biological material orbiological sample containing the nucleic acid was obtained as discussedsupra.

The present teachings are also directed to kits for extraction ofnucleic acid from a matrix, including, but not limited to, a biologicalmaterial that is calcified or otherwise hardened to a solid state, anadhesive, or sticky materials thought to contain nucleic acid and denimthought to contain nucleic acid. Such kits can be utilized forarcheological, criminal, forensic, human identification, and researchthat utilize the methods described above. In some embodiments, a basickit can comprise a container having a lysis solution capable ofreleasing nucleic acid from a matrix, an adhesive and/or sticky materialor denim material. A kit can also optionally comprise instructions foruse. The kit can also comprise other optional kit components, such as,for example, a solution comprising a polymer and detergent, magneticallyattractable particles, one or more wash solutions, a nucleic acidelution solution, a nucleic acid extraction control, a magnetic device,and a protocol and manual to educate the user and limit error in use.The amounts of the various reagents, tubes and magnetic device(s) in thekits also can be varied depending upon a number of factors, such as theoptimum purification and/or sensitivity of the process. It is within thescope of these teachings to provide test kits for use in manualapplications or test kits for use with automated sample preparation,reaction set-up, detectors, or analyzers.

Those in the art understand that the detection techniques employed aregenerally not limiting. Rather, a wide variety of nucleic acidextraction means are within the scope of the disclosed compositions,methods, and kits, provided that the material being analyzed containsnucleic acid or has nucleic acid adhered to the material.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are made only by way of example and are not intended tolimit the scope of the invention. What has been disclosed herein hasbeen provided for the purposes of illustration and description. It isnot intended to be exhaustive or to limit what is disclosed to theprecise forms described. Many modifications and variations will beapparent to the practitioner skilled in the art. What is disclosed waschosen and described in order to best explain the principles andpractical application of the disclosed embodiments of the art described,thereby enabling others skilled in the art to understand the variousembodiments and various modifications that are suited to the particularuse contemplated. It is intended that the scope of what is disclosed bedefined by the following claims and their equivalence.

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLES

Those having ordinary skill in the art will understand that manymodifications, alternatives, and equivalents are possible. All suchmodifications, alternatives, and equivalents are intended to beencompassed herein.

The following procedures are representative of reagents and proceduresthat can be employed for the extraction of nucleic acids from solidbiological matrices and adhesive substrates suspected of containingnucleic acid.

I. Sample Pre-Treatment

Bone and tooth samples were cleaned using mild detergent solution,overnight air drying, and removing the exterior layer using a sandingstone attached to a Dremel tool (Dremel, Madison, Wis.). Larger piecesof bones were chopped into small pieces. Pieces of bone and tooth werepulverized using a fresh or cleaned grinder (e.g., a Cryo-mill, BioSpectProducts, Bartlesville, Okla.). Pulverized bone or tooth power wasstored at room temperature.

Smoked cigarette butts were stored at room temperature. The filtermaterial was removed and the first 5 mm of the bottom portion of thepaper, which comes into contact with skin (i.e., finger and mouth) wascut into three pieces.

Chewed chewing gum samples were stored at room temperature. The gum wasflattened (˜3 mm thick) in a clean Petri dish. The lid was closed andthe Petri dish was stored at −80° C. for several hours for hardening.The hardened gum was cut into 8 equal sized portions. One portion of thegum was used for extraction of DNA.

Tape lift samples from various sources were prepared. From each sample,a 1.5 cm×2.5 cm piece of tape (containing the biological sample) wasused for processing. The blood or saliva was dry before removing by atape lift procedure and then adding the lysis reagent solution. For thetape+biological fluid samples, 0.5-5 μL of blood or saliva was addeddirectly to the adhesive side of the tape. For the tape lift samples, 2μL of human blood or 5 μL of saliva was added to a glass slide. Once thebiological material was dry, the biological material was tape lifted.Tape lift samples containing skin cells were also collected from eitherthe inside of a shirt collar or the inside of a shirt cuff from wornshirt samples.

Denim fabric samples (non-black denim) were prepared. 1 μL of blood inreplicates of 4 were obtained using a 5 mm punch where blood had beenapplied and allowed to air dry. The lysis reaction with theTris-Cl-buffered lysis reagent solution was carried out in a LySep™column (Applied Biosystems) in a 500 μL volume in the presence of 3 μLreducing agent (1 M) and 3 μL 20 mg/mL enzyme. The column was incubatedfor 40 min at 560 C on a thermal mixer. DNA extraction was done usingthe AutoMate Express™ (Applied Biosystems) following the manufacturersrecommended protocol. DNA quantification was done using Quantifiler® Duokit (Applied Biosystems) following the manufacturer's instructions. STRprofiling was performed using the Identifiler Kit following themanufacture's instructions and CE analysis was performed on the AppliedBiosystems 3130xl following the manufacture's instructions.

Denim fabric samples prepared with 20 μL of buccal epithelial cells in 6replicates were analyzed as described for blood on denim. Blood on blackdenim was 2 μL of blood in 3 replicates and was analyzed as describedfor denim above.

II. Preparation of Lysis Reagent Solution

Sufficient quantity of Lysis solution was prepared to process a numberof samples in duplicate or triplicate. The solution was freshly preparedby mixing 220 μL of 250-500 mM of chelating agent, pH 7.9 to 8.5containing 1% w/v of at least one detergent to which was added 0.5-1mg/mL enzyme and freshly prepared reducing agent in DNase free water ata final concentration of 10-100 mM. The lysis mixtures were incubatedwith or without shaking at a temperature in the range of approximately50-70° C. for a period of 90 minutes to 2 hours. Table 7 illustrateslysis solutions that can be used to extract nucleic acid from solidbiological materials and adhesive materials containing or thought tocontain biological samples containing nucleic acid.

III. Preparation of Tris-Cl-Buffered Lysis Reagent Solution

Sufficient quantity of Tris-Cl-buffered lysis reagent solution wasprepared to process a number of samples in duplicate, triplicate, or6-replicates. The solution was freshly prepared by mixing 220 μL of250-1000 mM of chelating agent, pH 7.9 to 8.5 containing 0.5-3% w/v ofat least one detergent to which was added 0.5-50 mg/mL enzyme andfreshly prepared reducing agent in DNase free water at a finalconcentration of 10-100 mM. The lysis mixtures were incubated with orwithout shaking at a temperature in the range of approximately 50-70° C.for a period of 90 minutes to 2 hours.

TABLE 7 Lysis Solutions Component Formulation I Formulation IIFormulation III Formulation IV Formulation V Formulation VI FormulationVII EGTA 250 to 500 mM 0 250 to 500 mM 0 250 to 500 mM 250 to 500 mM 250to 500 mM EDTA 0 250 to 500 mM 0 250 to 500 mM 0 0 0 DTT 10 to 100 mM 10to 100 mM 10 to 100 mM 10 to 100 mM 10 to 100 mM 10 to 100 mM 10 to 100mM NLS 0.5 to 3% w/v 0.5 to 3% w/v 0 0 0.5 to 3% w/v 0 0 Sodium 0 0 0.5to 3% w/v 0.5 to 3% w/v 0.5 to 3% w/v 0 0.5 to 3% w/v Deoxycholte SDS 00 0 0 0 0.1 to 0.5% w/v 0 Sodium 0 0 0 0 0 50 to 150 mM 0 Citrate TritonX 100 0 0 0 0 0 0.1 to 0.3% v/v 0 NP-40 0 0 0 0 0 0 0.1 to 0.3%Proteinase K 0.5 to 1 mg/mL 0.5 to 1 mg/mL 0.5 to 1 mg/mL 0.5 to 1 mg/mL0.5 to 1 mg/mL 0.5 to 1 mg/mL 0.5 to 1 mg/mL

TABLE 8 Wash Solutions: Wash Buffer A Final Wash Buffer B FinalComponent Concentration Concentration 10 mM Tris-Cl, pH 8.0   4 mM 0.1mM EDTA 0.04 mM 95% Ethanol  70% 60% Sodium deoxycholate (DSD) 0.5%Final Volume 100 mL 100 mLIII. Extraction of Nucleic Acid from Solid Biological Samples

230 μL of freshly prepared Lysis solution was added to 50-200 mg of boneor 10 mg of tooth powder, in a 2.0 mL screw cap tube. The lid was closedand the tube was incubated at 56° C. in an Eppendorf® Thermomixer R(Eppendorf North America, Westbury, N.Y.), at 1100 rpm for 2 hours. Atthe end of incubation, the tube was centrifuged at 10,000×g for 5 minand clear supernatant/lysate (maximal of 210 μL) was transferred to afresh 1.5 mL microfuge tube containing 300 μL of PrepFiler™ Lysis Bufferand processed for isolation of DNA according to the manufactures largevolume protocol. Basically, 15 μL of PrepFiler™ Magnetic particles and300 μL 99.5% Isopropanol is added followed by gentle mixing. Theparticles are was twice with PrepFiler™ Wash Buffer A using 600 μL andthen 300 μL of Buffer A followed by one wash with 300 μL PrepFiler™ WashBuffer B. The nucleic acid is then eluted into 50 μL of PrepFiler™Elution Buffer as outlined in the PrepFiler Manual and PrepFilerAutomated Forensic DNA Extraction Kit or the PrepFiler Manual Kit UserGuide (P/N 4390932-01).

IV. Extraction of Nucleic Acid from Adhesive Materials

230 μL of freshly prepared Lysis solution was added to the adhesivecontaining samples prepared as described above in a 1.5 mL microfugetube. The lid was closed and the tube was incubated at 56° C. in anEppendorf Thermomixer R at 750 rpm for 40 min. At the end of incubation,the contents were transferred to a filter column (Axygen Scientific,Inc., Union City, Calif.). The supernatant/lysate was separated from thesubstrate by centrifuging the filter column for 2 min at 16,110×g. 300μL of PrepFiler™ Lysis Buffer was added to the clear supernatant/lysateand processed for isolation of DNA according to the manufacturesprotocol.

V. Extraction of Nucleic Acid from Denim Materials

500 μL of freshly prepared Lysis solution was added to the biological(fluid) containing samples prepared as described above in a 1.5 mL LySeptube. The lid was closed and the tube was incubated at 56° C. in anEppendorf Thermomixer R at 750 rpm for 40 min. The supernatant/lysatewas separated from the substrate by centrifuging the LySep tube for 2min at 16,110×g. 300 μL of PrepFiler™ Lysis Buffer was added to theclear supernatant/lysate and processed for isolation of DNA according tothe manufactures protocol.

VI. Isolation of DNA

DNA from the lysate was isolated by following the large-sample protocol(for use with 500 μL of PrepFiler™ lysis buffer) as described by themanufacturer (Applied Biosystems, PrepFiler™ Forensic DNA Extraction KitUser Guide. PN 4390932 Rev. B, (2008)).

VII. Human DNA Quantification

The quantity of DNA was determined by Quantifiler® or Quantifiler® DuoHuman DNA Quantification Kit (Applied Biosystems, Foster City, Calif.)using 2 μL of the DNA extract as described in the User's Manual (AppliedBiosystems, Quantifiler® Human DNA Quantification Kit and Quantifiler® YHuman Male DNA Quantification Kit. User's Manual. PPN 4344790 Rev. B,(2003)). PCR was performed on the 7500 Real-Time PCR System and the datawere analyzed using 7500 System SDS Software v1.2.3 (Applied Biosystems,Foster City, Calif.).

VIII. Short Tandem Repeat (STR) Analysis

The DNA extracts obtained from the biological samples using PrepFiler™Forensic DNA Extraction Kit were amplified for Identifiler® kit analysisusing the procedure described in the User's Manual (Applied Biosystems,AmpFiSTR® Identifiler® PCR amplification kit. User's Manual. PPN 4323291Rev.B, (2001)). The amplified products were analyzed on a 3100 or 3130xlGenetic Analyzer (Applied Biosystems) using GeneMapper® ID Softwarev3.2.1 as described in the User Guides (Applied Biosystems, “ABI PRISM®3100 Genetic Analyzer and ABI PRISM® 3100-Avant Genetic Analyzer. UserReference Guide” PN 4335393. RevA, (2002), Applied Biosystems,GeneMapper® ID software for human identification applications).

Although the present disclosure is described with respect to variousembodiments and examples, various modifications may be made withoutdeparting from the spirit and scope of the invention.

1-12. (canceled)
 13. A method of making a product, wherein the productcomprises a nucleic acid, comprising: a) incubating a solid in a lysatesolution, the solid comprising the nucleic acid, wherein the lysatesolution comprises a detergent, a chelating agent, a reducing agent, andan enzyme; b) extracting a supernatant, wherein the supernatantcomprises the nucleic acid extracted from the solid.
 14. (canceled) 15.The method of claim 13, wherein the solid is a biological material, oran adhesive material or substrate.
 16. The method of claim 15, whereinthe biological material is selected from calcified tissue, bone,cartilage, ligament, tendon, and tooth.
 17. The method of claim 15,wherein the adhesive material comprises a biological sample.
 18. Themethod of claim 17, wherein the biological sample is embedded in theadhesive material substrate.
 19. The method of claim 18, wherein theadhesive material is selected from chewing gum, cigar butt, cigarettebutt, adhesive film, adhesive label, adhesive paper, adhesive skinpatch, envelope, stamp, a fingerprint tape lift, and adhesive tape. 20.The method of claim 19, wherein the adhesive skin patch is selected fromthe group consisting of electronic electrode, transferable tattoo,transdermal chemical substance patch and wound care dressing. 21.(canceled)
 22. The method of claim 13, wherein the detergent is selectedfrom one or more of N-lauroyl sarcosine, sodium deoxycholate, CTAB,dodecyl β-D-maltoside, nonanoyl-N-methylglucamide, polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodium dodecyl sulfate. 23.The method of claim 13, wherein the chelating agent comprises at leastone of ethylene glycol tetraacetic acid (EGTA), ethylene diaminetetraacetic acid (EDTA) and citric acid.
 24. The method of claim 13,wherein the reducing agent comprises at least one oftris(2-carboxyethyl)phosphine (TCEP) dithioerythritol (DTE),dithioerythritol (DTE), and dithiothreitol (DTT).
 25. The method ofclaim 13, wherein the enzyme comprises one or more of caspase,chymotrypsin, pepsin, proteinase K, thrombin, Staphylococcus V8protease, pronase, papain, Bacillus sp. E1A protease, and trypsin andcombinations thereof.
 26. The method of claim 13, further comprising thestep of shaking the incubating solution prior to extracting thesupernatant product. 27-50. (canceled)
 51. A method of separating anucleic acid from a solid sample, comprising: (a) lysing the solidsample in a lysis solution, thereby forming a mixture comprising thenucleic acid, wherein the lysate solution comprises a detergent, achelating agent, a reducing agent, and an enzyme; (b) centrifuging themixture, wherein the nucleic acid is thereby separated from the solidsample. 52-53. (canceled)
 54. The method of claim 53, wherein the solidis a biological material selected from bone, cartilage, ligament,tendon, and tooth.
 55. The method of claim 53, wherein the solid is anadhesive material that comprises a biological sample.
 56. (canceled) 57.The method of claim 55, wherein the adhesive material is selected fromchewing gum, cigar butt, cigarette butt, adhesive film, adhesive label,adhesive paper, adhesive skin patch, envelope, stamp, a fingerprint tapelift, and adhesive tape. 58-59. (canceled)
 60. The method of claim 51,wherein the detergent is selected from one or more of N-lauroylsarcosine, sodium deoxycholate, CTAB, dodecyl β-D-maltoside,nonanoyl-N-methylglucamide, polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodium dodecyl sulfatewherein the chelating agent comprises at least one of ethylene glycoltetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA) andcitric acid, wherein the reducing agent comprises at least one oftris(2-carboxyethyl)phosphine (TCEP) dithioerythritol (DTE), anddithiothreitol (DTT), and wherein the enzyme comprises one or more ofcaspase, chymotrypsin, pepsin, proteinase K, thrombin, Staphylococcus V8protease, pronase, papain, Bacillus sp. E1A protease, and trypsin andcombinations thereof. 61-67. (canceled)
 68. A method of identificationcomprising: (a) lysing a sample in a lysis solution, thereby forming alysate comprising a nucleic acid from the sample, wherein the lysatesolution comprises a detergent, a chelating agent, a reducing agent, andan enzyme; (b) extracting the lysate, wherein the nucleic acid is in thelysate; (c) analyzing the nucleic acid; and (d) identifying a source ofthe nucleic acid of the sample based on the nucleic acid analysis. 69.The method according to claim 68, wherein the sample is a fingerprinttape lift.
 70. (canceled)
 71. The method of claim 68, wherein thedetergent is selected from one or more of N-lauroyl sarcosine, sodiumdeoxycholate, CTAB, dodecyl β-D-maltoside, nonanoyl-N-methylglucamide,polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodiumdodecyl sulfate wherein the chelating agent comprises at least one ofethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraaceticacid (EDTA) and citric acid, wherein the reducing agent comprises atleast one of tris(2-carboxyethyl)phosphine (TCEP) dithioerythritol(DTE), and dithiothreitol (DTT), and wherein the enzyme comprises one ormore of caspase, chymotrypsin, pepsin, proteinase K, thrombin,Staphylococcus V8 protease, pronase, papain, Bacillus sp. E1A protease,and trypsin and combinations thereof. 72-100. (canceled)